Organelle identity and development rely on a complex set of intracellular protein trafficking systems that mediate the specific targeting of nucleus-encoded proteins to their proper subcellular compartment. Not only must these trafficking systems maintain a high degree of specificity, they often must adapt to accommodate dramatic changes in the levels and composition of trafficking substrates imposed by developmental, stress and physiological events. Chloroplasts have evolved from the original endosymbiont to perform specialized functions in different tissues and cell types, giving rise to a diverse group of inter-related organelles called plastids. Plastid function and development rely on the coordinated expression and post-translational import of ~3000 different nucleus-encoded proteins. As such, plastids are an ideal model for studies of preprotein recognition and membrane translocation, and thereby, contribute to our knowledge of the basic processes of intracellular protein targeting and organelle biogenesis. A major aim of this proposal is to define the determinants and molecular interactions that mediate recognition of the targeting signals (transit peptides) of chloroplast preproteins by the TOC GTPase receptors at the chloroplast surface, and understand the process by which transit peptide recognition is coupled to GTP-dependent transfer of the preprotein into the membrane channel at the outer membrane of the organelle. The proposed roles of the TOC GTPases are reminiscent of the roles of the signal recognition particle (SRP/Ffh) and SRP receptor (SR/FtsY) in preprotein targeting to the ER and bacterial cytoplasmic membranes. In this context, the TOC translocon represent a novel, but analogous example of the GTP-regulated switches that regulate numerous intracellular protein targeting pathways. Our studies also suggest that multiple TOC pathways have evolved to balance protein targeting with the changes in gene expression that accompany developmental, physiological and stress events in cells. We will use the chloroplast system to understand the network of processes that 1) control the overall levels of organellar proteins expressed from the nucleus, 2) maintain the stichiometry of multi-protein complexes that contain both organelle and nucleus-encoded subunits, 3) respond to organelle status or dysfunction and, 4) coordinate changes in protein profiles during developmental transitions from one organelle type to another. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand the molecular mechanism by which proteins are targeted to their proper cellular compartment or organelle using chloroplasts as a model system. The facile biochemical and molecular genetic tools available to study protein targeting in Arabidopsis thaliana makes this an ideal system to define general principles of protein targeting that apply to organelle systems in other eukaryotic cells and in bacteria. As such, these studies will contribute to our understanding of a fundamental process that is required for normal cellular function.